Dengue has evolved from a virus primarily maintained in canopy-dwelling Aedes mosquitoes and lower primates to an Ae aegypti-human-Ae. aegypti cycle. Dengue fever has been reported since the 1800s, but these cases have been sporadic with low mortality rates. Since the 1950s, successive epidemics have increased in frequency, size, and severity as both human and insect mobility has increased. There are four well-characterized dengue serotypes, DEN1-4, that are currently circulating globally. In some areas where only one dengue serotype circulated, now all four are found. The most serve manifestations of disease are associated with Den 2 and 3, although all serotypes can cause mortality. Even within one serotype, viral genotype has been shown to alter transmission and pathogenicity. Successive infection by heterologous serotypes leads to an increased severe disease manifestation. The studies proposed here are on DEN1-2 and 4 that have been co-circulating since 1980 in Puerto Rico. There has been yearly endemic transmission of the virus punctuated by five epidemics from 1980-1998. Current data indicate that epidemics can be dominated by one or multiple serotypes and that the relative importance of serotypes can shift between epidemics. The basic idea of this proposal is to trace and identify the evolutionary change in the Dengue genome that allows for changes in transmission and disease severity. There are several hypotheses that could account for this: 1) the immigration of new dengue genotypes into stable dengue populations; 2) expansion of specific genotypes in the quasi-species due to expanded host range; and 3) co- circulation and potential recombination of multiple dengue serotypes. The studies proposed here will address these possibilities and try to correlate specific amino acid changes (or multiple sets thereof) with pathogenesis and/or epidemic potential.